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Drosophila Embryo and the Mechanisms That Generate Tissue-Specific Outputs of Hedgehog Signaling Brian Biehs, Katerina Kechris*, Songmei Liu and Thomas B

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Drosophila Embryo and the Mechanisms That Generate Tissue-Specific Outputs of Hedgehog Signaling Brian Biehs, Katerina Kechris*, Songmei Liu and Thomas B RESEARCH ARTICLE 3887 Development 137, 3887-3898 (2010) doi:10.1242/dev.055871 © 2010. Published by The Company of Biologists Ltd Hedgehog targets in the Drosophila embryo and the mechanisms that generate tissue-specific outputs of Hedgehog signaling Brian Biehs, Katerina Kechris*, SongMei Liu and Thomas B. Kornberg† SUMMARY Paracrine Hedgehog (Hh) signaling regulates growth and patterning in many Drosophila organs. We mapped chromatin binding sites for Cubitus interruptus (Ci), the transcription factor that mediates outputs of Hh signal transduction, and we analyzed transcription profiles of control and mutant embryos to identify genes that are regulated by Hh. Putative targets that we identified included several Hh pathway components, mostly previously identified targets, and many targets that are novel. Every Hh target we analyzed that is not a pathway component appeared to be regulated by Hh in a tissue-specific manner; analysis of expression patterns of pathway components and target genes provided evidence of autocrine Hh signaling in the optic primordium of the embryo. We present evidence that tissue specificity of Hh targets depends on transcription factors that are Hh- independent, suggesting that ‘pre-patterns’ of transcription factors partner with Ci to make Hh-dependent gene expression position specific. KEY WORDS: Drosophila, Hedgehog, Cubitus interruptus INTRODUCTION Pleiotropic phenotypes result from loss or inactivation of Hh Hedgehog (Hh) is a secreted signaling protein that regulates the pathway components, suggesting that the pathway is similarly growth and patterning of many organs. First identified because of constituted in the affected tissues (reviewed by McMahon et al., its roles in Drosophila embryonic and imaginal disc development, 2003). Ci is an essential core pathway component (Alexandre et al., it is now understood to be essential to most organs in Drosophila 1996; Forbes et al., 1993) and is stabilized by Hh signal and higher vertebrates. Despite its systemic importance, Hh transduction (Aza-Blanc et al., 1997; Ruel et al., 2003). Expression signaling is local and is dependent upon Hh that is expressed and of ptc (Forbes et al., 1993; Ingham, 1991; Tabata and Kornberg, secreted by discrete sets of cells in each tissue that it regulates. 1994) and roadkill (rdx) (Kent et al., 2006; Zhang et al., 2006) are In each context, organ-specific programs of gene expression, also hallmarks of Hh action. Many of the pathway components are morphogenesis and cell-cycle regulation depend upon Hh evolutionarily conserved in higher vertebrates and most of these regulation. The mechanism that endows Hh signaling with tissue homologs have been found to play similar roles in the specificity has not been fully elucidated. corresponding Hh pathways (reviewed by Varjosalo and Taipale, Hh signaling has been most thoroughly analyzed in the 2008). The deep homology extends to the three Gli transcription Drosophila wing imaginal disc, where Hh is expressed in posterior factors (Orenic et al., 1990), homologs of Ci that share a consensus compartment cells. Signaling to adjacent anterior cells begins when binding sequence (TGGGTGGTC) (Hallikas et al., 2006; Kinzler Hh engages the participation of two membrane proteins in target and Vogelstein, 1990; Pavletich and Pabo, 1993). cells: Patched (Ptc) and Smoothened (Smo). Hh activates Smo As with all signaling systems that employ a common mechanism (Stone et al., 1996; Taipale et al., 2002) and initiates the of signal transduction to regulate a variety of target tissues, the transformation of a complex of proteins that includes an inactive question arises of how tissue-specific responses are induced. For the form of the transcription factor Ci (Jia et al., 2003; Lum et al., Hh pathway, the question of specificity also applies to CiAct and 2003; Ruel et al., 2003). Ci mediates most, and perhaps all, of the CiRep, which regulate expression of targets in the on and off states, output of the Hh pathway (Alexandre et al., 1996; Méthot and respectively. Analysis of the imaginal disc enhancer of Basler, 2001). In the absence of Hh, cleavage of Ci generates a decapentaplegic (dpp) revealed that both CiAct and CiRep can bind truncated peptide that functions as a transcriptional repressor to, and regulate, the same binding site sequence (Müller and Basler, (CiRep) (Aza-Blanc et al., 1997). The presence of Hh reduces CiRep 2000). However, we do not yet know whether such shared access is and enhances production of a transcriptional activator form (CiAct) a feature of all Ci binding sites and, if it is, how the distinct activator (Aza-Blanc et al., 1997; Méthot and Basler, 1999). and repressor functions are realized in the context of their common sites. Possible mechanisms include differential affinity, input from linked cis-regulatory elements or participation of co-factors (Müller and Basler, 2000). Although support for the latter might come from Cardiovascular Research Institute and Department of Biochemistry and Biophysics, the observation that full activation of the dpp heldout enhancer University of California, San Francisco, CA 94143-2711. requires both Hh signaling and the wing ‘selector’ protein Vestigial *Present address: Colorado School of Public Health, University of Colorado Denver, (Hepker et al., 1999), the mechanisms that determine tissue-specific 13001 E. 17th Place B-119, Aurora, CO 80045 activation of other Hh targets remain unidentified. Recent studies †Author for correspondence (tkornberg(at)ucsf.edu) that used chromatin binding to identify target sequences for mouse Accepted 16 September 2010 Gli1 and Gli3 proteins in neural tissues and whole limb buds, DEVELOPMENT 3888 RESEARCH ARTICLE Development 137 (22) respectively (Vokes et al., 2007; Vokes et al., 2008), identified many transgenes. DNA (2.5 mg) was ethanol-precipitated, digested with DpnI and novel Gli-responsive cis-regulatory elements. Characterization of ligated to annealed adaptor oligos for 2 hours at 16°C (5Ј-CTAA - these elements led to the conclusion that the Gli1 activator and the TACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGA-3Ј and Gli3 repressor recognize and regulate common sequences. 5Ј-TCCTCGGCCG-3Ј). After heat inactivation, the ligation mixture was The number of known Hh targets in Drosophila is small. The digested with DpnII, which cuts unmethylated DNA. PCR amplification of work described here was undertaken to obtain a better understanding Dam-methylated DNA was carried out using the PCR Advantage Mix (Clontech) and the primer 5Ј-GGTCGCGGCCGAGGATC-3Ј. Labeling of targets and to investigate the mechanism of Ci action and and hybridizations were performed by NimbleGen Systems (Reykjavík, specificity. We employed expression array assays to identify genes Iceland). The amplified methylated fragments were labeled with Cy3 or that have Hh-dependent expression in embryos, and chromatin- Cy5 in a dye-swap configuration to eliminate bias and were hybridized to binding assays to identify genes linked to genomic regions that are NimbleGen Systems’ whole genome tiling arrays consisting of 375,000 recognized by CiAct and CiRep. Putative targets were selected that 60mer probes spaced ~300 bp apart (Choksi et al., 2006). Three replicates bind both Ci forms and are Hh-dependent. Characterization of of control (Dam alone) and experimental (DamCiAct and DamCiRep) several novel targets highlighted two key aspects of Hh signaling. samples were analyzed. Raw intensity data were analyzed as described We found evidence for autocrine signaling and also found that, with below. the exception of several genes that encode core components of the All analyses were performed in R v2.7.1 (R Development Core Team, Hh signal transduction pathway, all targets are expressed in restricted http://www.R-project.org/) unless otherwise stated. At each tiling array feature, log ratios between the experimental and control samples were domains within Hh-responsive tissues. For Ecdysone-inducible gene calculated and normalized using ‘limma’ (Smyth, 2005), applying ‘loess’ L2 (ImpL2), a novel Hh-regulated gene that is expressed in the and ‘Aquantile’ for normalization within arrays and between arrays, tracheal primordium, we show that expression is dependent upon the respectively (Wormald et al., 2006). To predict binding regions, each transcription factor Trachealess (Trh), expression of which is not feature was tested for significant positive intensity using a one-sided t-test. dependent upon Hh, and that ImpL2 expression is restricted to only Because sample size was small (n3), information was pooled from all a portion of the cells that express Trh. Expression of this Hh target features to obtain more stable variance estimates for the t-statistic using the is, therefore, specified by the combined activities of Hh-dependent limma package. To identify genomic regions of high signal intensity, P- Ci and Hh-independent Trh. values for consecutive features were ‘averaged’ using meta-analysis based on combining P-values (for details, see Kechris et al., 2010). Because of MATERIALS AND METHODS the large number of significance tests, P-values were corrected for false Fly stocks and crosses discovery rate (FDR) control (Benjamini et al., 2001) with window size set Homozygous null mutant embryos lacking ptc or hh function were at four. Binding regions were created by scanning features as they are generated from the following alleles: hh13c, hhAC, ptcB98 and ptc13C. ci-null ordered along each chromosome. If a feature had an adjusted P-value Rep Act embryos were generated from a cross of ciRES (Méthot and Basler, 1999) below the set FDR cutoff (0.02 for DamCi ; 0.001 for DamCi ), a new in a ci-null background (ci94/ci94). smo germline clones were generated binding region was formed. The next feature passing the cutoff in the linear from a cross of y w hs-FLP/+; smoQ FRT40A/CyO females to +/Y; OvoD1, chromosome was then evaluated. This procedure was continued in a step- FRT40A/CyO males (Chou and Perrimon, 1996). Embryos and first instar wise fashion. progeny were heat shocked daily at 37°C for 1 hour prior to eclosion. UAS- m1-m4 Act Overlap of DamID protected regions Ci (Ci ) was obtained from S.
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